Small Dense

7/31/2019 Small Dense

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Elizabeth Teng Leary, Ph.D.

Chief Scientific OfficerPacific Biometrics, Inc.

July 26, 2006 AACC Chicago

Small Dense LDL


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Small LDL Correlates with CVD Risks

Relative risk is 4.5 for CAD and 7 for MI when small,dense LDL > 100mg/dL (Griffin B A ,et al, Atherosclerosis

1994).

People with predominance of small, dense LDL (i.e.,

Pattern B patients) have a 3-fold increased risk of

myocardial infarction (Austin M A ,et al, JAMA1988).


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Reports on sd LDL at ISA 2006

Small dense LDL are strongly correlated with increasedCHD risk especially the proportion and absolute levels.[GGE] (B. Lamarche, Quebec, Canada)

Small LDL has prolonged plasma residence timecompared to large LDL as shown by stable isotope kinetics(B. Lamarche, Quebec, Canada)

Ashkenazi Jews of exceptionoal longevity and theiroffsprings possess larger LDL and HDL particles andincreased HDL-C. [NMR] (N. Barzilari, NY, USA)


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Reports on sdLDL at ISA 2006

In a free-living population with no sign or history of CVDnor on any lipid-lowering therapy, LDL size and HDL-C arethe only independent predictors of carotid IMT. Only LDL-Ccarried in the remnant and sdLDL fraction is stronglycorrelated with degree of IMT. [ UC] (A. Zambon, Milan, Italy)

In women with angiogrpahically confirmed CAD, bothsmall LDL and HDL phenotypes are predictive of increase inrisk. Small HDL alone has greater predictive value. [GGE]

(P. Blackburn, Quebec, Canada)


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Challenges of sd LDL Interpretation

Epidemiologic data in support of small LDL being anindependent risk factor is still lacking

Proper interpretation is hampered by the wide variety ofLDL subclass measurements used in clinical studies

Current LDL subclass measurements are based ondifferent principles and are non-standardized among orwithin technology and non-standardized in reporting

convention

Available methods are often technique demanding,require special equipment and may not be suitable for

routine use


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Lipoprotein Subclasses


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Definitions for small, dense LDL

Lipoproteins VLDL L LDL sd LDL * HDL

Diameter

(nm) 30 80 25.5 - 28.0 22.0 - 25.5 7 10

1.063 -

1.210

Density

(g/mL)


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Lipoprotein Subclass Measurements

Method Principle Reporting Availability

GGE Non-denaturing gradient gelSeparate by size in electrical field

Size, % fraction Home brew / proprietary

commercial lab

NMR Proton NMRSeparate by methyl group signal

Size and particle number;quantification by convolutedalgorithm

Proprietarycommercial lab

UC Sequential or density gradient UCSeparate by densityDirect component measurement

Size, lipid components; subclassquntification by convolutedalgorithm in sequential UC

Home brew /proprietarycommercial lab

PAGE Fixed concentration disk PAGESeparate by charge

Ave peak size, % fraction Home brew /Commerciallyavailable

HPLC Size exclusion chromatographySeparate by size

Direct component measurement

Peak size; lipid components;subclass quantification byconvoluted algorithm

Home brew /proprietarycommercial lab

Polyanion-cation

Precipitation

Heparin-Mg precipitation

Apo content, selective detergent and

enzymesDirect component measurement

Small dense LDL chol and othercomponents

Commerciallyavailable


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Grouping of patients with major particle size

Pattern A(individual w/ predominance

of large, buoyant LDL)

26.2 nm

25.1 nm

24.1 nm

Major peak of particle diametersAt 25.5nm or greater

Pattern B(individual w/ with predominance

of small, dense LDL)

25.0 nm

24.4 nm

Major peak of particle diametersAt less than 25.5nm


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sd LDL-C SEIKEN Features

Quantitatively measures small, dense LDL, result is

reported in mg/dL cholesterol

Procedure is simple, no special technique is required

No need for special equipment; only a microcentrifuge,water bath and chemistry analyzer

Test is completed within 60 min


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sd LDL-C SEIKEN Procedure

Step 1 : Sample pre-treatment

Separate small, dense LDL from large LDL, VLDL, IDL, CMwith heparin and Mg Pretreatment Solution

Centrifugation

tube

Samplecup 1,500~10,000 g

1 min

37o

C

10 min

Large LDL, VLDL &CM are trapped bythe filter

Collect the filtrate as thesample for the next step[sd LDL + HDL]

200 uLPretreatment

solution200 uL Sample


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sd LDL-C SEIKEN Procedure

Assay Step-2 : Cholesterol Analysis in Two-reagent Assay

Decomposition of HDL (by R-1)

Cholesterol 2H2O2 2H2O + O2CatalaseEsterase & Oxidase

Surfactant A (specific to HDL)

Determination of sLDL-C (by R-2)

Surfactant B

Cholesterol 2H2O2 Purple-red colorPODEsterase & Oxidase


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sd LDL-C Sample Stability

Maximum Stability:

In whole blood up to 6 h at 8 C or 25 C

In serum or EDTA plasma

- 2 d at 8 C

- 7 d at -20 C

- 12 mo at -80 C

In filtrate from pretreatment up to 7-15 d


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0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

0 1 2 3 4 7

Days at 4 C

sdLDL

(mg/dL)

QC 1

S1

S2

S3

S4

S5

S6

S7

S8

S9

QC 2

QC PBI

Stability of sd LDL Filtrates at 4C


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sd LDL-C Precision

151610n

8.24.920.2CV%

5.71.60.79SD

69.832.33.9Mean

QC Kit HQC Kit LPBI LQC Name

Between Run Precision202020n

4.62.313.8CV%

3.280.770.37SD

71.433.72.7Mean

QC Kit HQC Kit LPBI LQC Name

Within Run Precision


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sd LDL-C SEIKEN

Correlation with ultracentrifugation method

y = 0.95x - 0.38

R = 0.910

0

20

40

60

80

100

0 20 40 60 80 100

Ultracentrifugation mg/dL

DenkasdLDLassay

mg/dL

64 Samples from healthy people, CAD and Diabetic patients


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Summary

Current LDL subfractionation methods are based on avariety of technologies and principles. They are oftentechnically demanding, laborious or proprietary thus not

suitable for use in the routine laboratory

Lack of consistency and standardization of LDL subclassmeasurements prevents true comparison, limitingopportunity for greater exploration of small LDL as a markerof CVD

Most representative small dense LDL assay for CVD riskremains to be determined

Denka sdLDL is a promising new tool suitable for the

routine lab that may assist in investigating this importantarea


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Further Considerations of Denka sdLDL Assay

Further streamlining of procedure to improve robustness

Establish long-term reagent lot-to-lot consistency tosupport clinical diagnostic use

Evaluate specificity in various phenotypes, clinicalsamples and in the presence of potential interferences

Investigate best representative marker of small dense

LDL - chol? total or free? apo B? phospholipid?


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Small Dense

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